14.
Nueda, M. J.; Conesa, A.; Westerhuis, J. A.; Hoefsloot, H. C. J.; Smilde, A. K.; Talon, M.; Ferrer, A.
Discovering gene expression patterns in time course microarray experiments by ANOVA-SCA Journal Article
In: BIOINFORMATICS, vol. 23, no. 14, pp. 1792-1800, 2007, ISSN: 1367-4803.
@article{ISI:000249248300011,
title = {Discovering gene expression patterns in time course microarray
experiments by ANOVA-SCA},
author = { M. J. Nueda and A. Conesa and J. A. Westerhuis and H. C. J. Hoefsloot and A. K. Smilde and M. Talon and A. Ferrer},
url = {http://dx.doi.org/10.1093/bioinformatics/btm251},
doi = {10.1093/bioinformatics/btm251},
issn = {1367-4803},
year = {2007},
date = {2007-07-01},
journal = {BIOINFORMATICS},
volume = {23},
number = {14},
pages = {1792-1800},
abstract = {Motivation: Designed microarray experiments are used to investigate the
effects that controlled experimental factors have on gene expression and
learn about the transcriptional responses associated with external
variables. In these datasets, signals of interest coexist with varying
sources of unwanted noise in a framework of (co)relation among the
measured variables and with the different levels of the studied factors.
Discovering experimentally relevant transcriptional changes require
methodologies that take all these elements into account.
Results: In this work, we develop the application of the Analysis of
variance-simultaneous component analysis (ANOVA-SCA) Smilde et al, Bioinformatics, (2005) to the analysis of multiple series time course
microarray data as an example of multifactorial gene expression
profiling experiments. We denoted this implementation as ASCA-genes. We
show how the combination of ANOVA-modeling and a dimension reduction
technique is effective in extracting targeted signals from data
by-passing structural noise. The methodology is valuable for identifying
main and secondary responses associated with the experimental factors
and spotting relevant experimental conditions. We additionally propose a
novel approach for gene selection in the context of the relation of
individual transcriptional patterns to global gene expression signals.
We demonstrate the methodology on both real and synthetic datasets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Motivation: Designed microarray experiments are used to investigate the
effects that controlled experimental factors have on gene expression and
learn about the transcriptional responses associated with external
variables. In these datasets, signals of interest coexist with varying
sources of unwanted noise in a framework of (co)relation among the
measured variables and with the different levels of the studied factors.
Discovering experimentally relevant transcriptional changes require
methodologies that take all these elements into account.
Results: In this work, we develop the application of the Analysis of
variance-simultaneous component analysis (ANOVA-SCA) Smilde et al, Bioinformatics, (2005) to the analysis of multiple series time course
microarray data as an example of multifactorial gene expression
profiling experiments. We denoted this implementation as ASCA-genes. We
show how the combination of ANOVA-modeling and a dimension reduction
technique is effective in extracting targeted signals from data
by-passing structural noise. The methodology is valuable for identifying
main and secondary responses associated with the experimental factors
and spotting relevant experimental conditions. We additionally propose a
novel approach for gene selection in the context of the relation of
individual transcriptional patterns to global gene expression signals.
We demonstrate the methodology on both real and synthetic datasets.
effects that controlled experimental factors have on gene expression and
learn about the transcriptional responses associated with external
variables. In these datasets, signals of interest coexist with varying
sources of unwanted noise in a framework of (co)relation among the
measured variables and with the different levels of the studied factors.
Discovering experimentally relevant transcriptional changes require
methodologies that take all these elements into account.
Results: In this work, we develop the application of the Analysis of
variance-simultaneous component analysis (ANOVA-SCA) Smilde et al, Bioinformatics, (2005) to the analysis of multiple series time course
microarray data as an example of multifactorial gene expression
profiling experiments. We denoted this implementation as ASCA-genes. We
show how the combination of ANOVA-modeling and a dimension reduction
technique is effective in extracting targeted signals from data
by-passing structural noise. The methodology is valuable for identifying
main and secondary responses associated with the experimental factors
and spotting relevant experimental conditions. We additionally propose a
novel approach for gene selection in the context of the relation of
individual transcriptional patterns to global gene expression signals.
We demonstrate the methodology on both real and synthetic datasets.
13.
Terol, J.; Conesa, A.; Colmenero, J. M.; Cercos, M.; Tadeo, F.; Agusti, J.; Alos, E.; Andres, F.; Soler, G.; Brumos, J.; Iglesias, D. J.; Gotz, S.; Legaz, F.; Argout, X.; Courtois, B.; Ollitrault, P.; Dossat, C.; Wincker, P.; Morillon, R.; Talon, M.
Analysis of 13000 unique Citrus clusters associated with fruit quality, production and salinity tolerance Journal Article
In: BMC GENOMICS, vol. 8, 2007, ISSN: 1471-2164.
@article{ISI:000244326200001,
title = {Analysis of 13000 unique Citrus clusters associated with fruit quality, production and salinity tolerance},
author = { J. Terol and A. Conesa and J. M. Colmenero and M. Cercos and F. Tadeo and J. Agusti and E. Alos and F. Andres and G. Soler and J. Brumos and D. J. Iglesias and S. Gotz and F. Legaz and X. Argout and B. Courtois and P. Ollitrault and C. Dossat and P. Wincker and R. Morillon and M. Talon},
url = {http://dx.doi.org/10.1186/1471-2164-8-31},
doi = {10.1186/1471-2164-8-31},
issn = {1471-2164},
year = {2007},
date = {2007-01-01},
journal = {BMC GENOMICS},
volume = {8},
abstract = {Background: Improvement of Citrus, the most economically important fruit
crop in the world, is extremely slow and inherently costly because of
the long-term nature of tree breeding and an unusual combination of
reproductive characteristics. Aside from disease resistance, major
commercial traits in Citrus are improved fruit quality, higher yield and
tolerance to environmental stresses, especially salinity.
Results: A normalized full length and 9 standard cDNA libraries were
generated, representing particular treatments and tissues from selected
varieties ( Citrus clementina and C. sinensis) and rootstocks ( C.
reshni, and C. sinenis x Poncirus trifoliata) differing in fruit
quality, resistance to abscission, and tolerance to salinity. The goal
of this work was to provide a large expressed sequence tag ( EST)
collection enriched with transcripts related to these well appreciated
agronomical traits. Towards this end, more than 54000 ESTs derived from
these libraries were analyzed and annotated. Assembly of 52626 useful
sequences generated 15664 putative transcription units distributed in
7120 contigs, and 8544 singletons. BLAST annotation produced significant
hits for more than 80% of the hypothetical transcription units and
suggested that 647 of these might be Citrus specific unigenes. The
unigene set, composed of similar to 13000 putative different
transcripts, including more than 5000 novel Citrus genes, was assigned
with putative functions based on similarity, GO annotations and protein
domains.
Conclusion: Comparative genomics with Arabidopsis revealed the presence
of putative conserved orthologs and single copy genes in Citrus and also
the occurrence of both gene duplication events and increased number of
genes for specific pathways. In addition, phylogenetic analysis
performed on the ammonium transporter family and glycosyl transferase
family 20 suggested the existence of Citrus paralogs. Analysis of the
Citrus gene space showed that the most important metabolic pathways
known to affect fruit quality were represented in the unigene set.
Overall, the similarity analyses indicated that the sequences of the
genes belonging to these varieties and rootstocks were essentially
identical, suggesting that the differential behaviour of these species
cannot be attributed to major sequence divergences. This Citrus EST
assembly contributes both crucial information to discover genes of
agronomical interest and tools for genetic and genomic analyses, such as
the development of new markers and microarrays.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Background: Improvement of Citrus, the most economically important fruit
crop in the world, is extremely slow and inherently costly because of
the long-term nature of tree breeding and an unusual combination of
reproductive characteristics. Aside from disease resistance, major
commercial traits in Citrus are improved fruit quality, higher yield and
tolerance to environmental stresses, especially salinity.
Results: A normalized full length and 9 standard cDNA libraries were
generated, representing particular treatments and tissues from selected
varieties ( Citrus clementina and C. sinensis) and rootstocks ( C.
reshni, and C. sinenis x Poncirus trifoliata) differing in fruit
quality, resistance to abscission, and tolerance to salinity. The goal
of this work was to provide a large expressed sequence tag ( EST)
collection enriched with transcripts related to these well appreciated
agronomical traits. Towards this end, more than 54000 ESTs derived from
these libraries were analyzed and annotated. Assembly of 52626 useful
sequences generated 15664 putative transcription units distributed in
7120 contigs, and 8544 singletons. BLAST annotation produced significant
hits for more than 80% of the hypothetical transcription units and
suggested that 647 of these might be Citrus specific unigenes. The
unigene set, composed of similar to 13000 putative different
transcripts, including more than 5000 novel Citrus genes, was assigned
with putative functions based on similarity, GO annotations and protein
domains.
Conclusion: Comparative genomics with Arabidopsis revealed the presence
of putative conserved orthologs and single copy genes in Citrus and also
the occurrence of both gene duplication events and increased number of
genes for specific pathways. In addition, phylogenetic analysis
performed on the ammonium transporter family and glycosyl transferase
family 20 suggested the existence of Citrus paralogs. Analysis of the
Citrus gene space showed that the most important metabolic pathways
known to affect fruit quality were represented in the unigene set.
Overall, the similarity analyses indicated that the sequences of the
genes belonging to these varieties and rootstocks were essentially
identical, suggesting that the differential behaviour of these species
cannot be attributed to major sequence divergences. This Citrus EST
assembly contributes both crucial information to discover genes of
agronomical interest and tools for genetic and genomic analyses, such as
the development of new markers and microarrays.
crop in the world, is extremely slow and inherently costly because of
the long-term nature of tree breeding and an unusual combination of
reproductive characteristics. Aside from disease resistance, major
commercial traits in Citrus are improved fruit quality, higher yield and
tolerance to environmental stresses, especially salinity.
Results: A normalized full length and 9 standard cDNA libraries were
generated, representing particular treatments and tissues from selected
varieties ( Citrus clementina and C. sinensis) and rootstocks ( C.
reshni, and C. sinenis x Poncirus trifoliata) differing in fruit
quality, resistance to abscission, and tolerance to salinity. The goal
of this work was to provide a large expressed sequence tag ( EST)
collection enriched with transcripts related to these well appreciated
agronomical traits. Towards this end, more than 54000 ESTs derived from
these libraries were analyzed and annotated. Assembly of 52626 useful
sequences generated 15664 putative transcription units distributed in
7120 contigs, and 8544 singletons. BLAST annotation produced significant
hits for more than 80% of the hypothetical transcription units and
suggested that 647 of these might be Citrus specific unigenes. The
unigene set, composed of similar to 13000 putative different
transcripts, including more than 5000 novel Citrus genes, was assigned
with putative functions based on similarity, GO annotations and protein
domains.
Conclusion: Comparative genomics with Arabidopsis revealed the presence
of putative conserved orthologs and single copy genes in Citrus and also
the occurrence of both gene duplication events and increased number of
genes for specific pathways. In addition, phylogenetic analysis
performed on the ammonium transporter family and glycosyl transferase
family 20 suggested the existence of Citrus paralogs. Analysis of the
Citrus gene space showed that the most important metabolic pathways
known to affect fruit quality were represented in the unigene set.
Overall, the similarity analyses indicated that the sequences of the
genes belonging to these varieties and rootstocks were essentially
identical, suggesting that the differential behaviour of these species
cannot be attributed to major sequence divergences. This Citrus EST
assembly contributes both crucial information to discover genes of
agronomical interest and tools for genetic and genomic analyses, such as
the development of new markers and microarrays.
12.
Williams, T. D.; Diab, A. M.; George, S. G.; Godfrey, R. E.; Sabine, V.; Conesa, A.; Minchin, S. D.; Watts, P. C.; Chipman, J. K.
In: ENVIRONMENTAL SCIENCE & TECHNOLOGY, vol. 40, no. 20, pp. 6479-6488, 2006, ISSN: 0013-936X.
@article{ISI:000241192600050,
title = {Development of the GENIPOL European flounder (Platichthys flesus)
microarray and determination of temporal transcriptional responses to
cadmium at low dose.},
author = { T. D. Williams and A. M. Diab and S. G. George and R. E. Godfrey and V. Sabine and A. Conesa and S. D. Minchin and P. C. Watts and J. K. Chipman},
url = {http://dx.doi.org/10.1021/es061142h},
doi = {10.1021/es061142h},
issn = {0013-936X},
year = {2006},
date = {2006-10-01},
journal = {ENVIRONMENTAL SCIENCE & TECHNOLOGY},
volume = {40},
number = {20},
pages = {6479-6488},
abstract = {We have constructed a high density, 13 270-clone cDNA array for the
sentinel fish species European flounder (Platichthys flesus), combining
clones from suppressive subtractive hybridization and a liver cDNA
library; DNA sequences of 5211 clones were determined. Fish were treated
by single intraperitoneal injection with 50 micrograms cadmium chloride
per kilogram body weight, a dose relevant to environmental exposures, and hepatic gene expression changes were determined at 1, 2, 4, 8, and
16 days postinjection in comparison to saline-treated controls. Gene
expression responses were confirmed by real-time reverse transcription
polymerase chain reaction (RT-PCR). Blast2GO gene ontology analysis
highlighted a general induction of the unfolded protein response, response to oxidative stress, protein synthesis, transport, and
degradation pathways, while apoptosis, cell cycle, cytoskeleton, and
cytokine genes were also affected. Transcript levels of cytochrome P450
1A (CYP1A) were repressed and vitellogenin altered, real-time PCR showed
induction of metallothionein. We thus describe the establishment of a
useful resource for ecotoxicogenomics and the determination of the
temporal molecular responses to cadmium, a prototypical heavy metal
pollutant.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We have constructed a high density, 13 270-clone cDNA array for the
sentinel fish species European flounder (Platichthys flesus), combining
clones from suppressive subtractive hybridization and a liver cDNA
library; DNA sequences of 5211 clones were determined. Fish were treated
by single intraperitoneal injection with 50 micrograms cadmium chloride
per kilogram body weight, a dose relevant to environmental exposures, and hepatic gene expression changes were determined at 1, 2, 4, 8, and
16 days postinjection in comparison to saline-treated controls. Gene
expression responses were confirmed by real-time reverse transcription
polymerase chain reaction (RT-PCR). Blast2GO gene ontology analysis
highlighted a general induction of the unfolded protein response, response to oxidative stress, protein synthesis, transport, and
degradation pathways, while apoptosis, cell cycle, cytoskeleton, and
cytokine genes were also affected. Transcript levels of cytochrome P450
1A (CYP1A) were repressed and vitellogenin altered, real-time PCR showed
induction of metallothionein. We thus describe the establishment of a
useful resource for ecotoxicogenomics and the determination of the
temporal molecular responses to cadmium, a prototypical heavy metal
pollutant.
sentinel fish species European flounder (Platichthys flesus), combining
clones from suppressive subtractive hybridization and a liver cDNA
library; DNA sequences of 5211 clones were determined. Fish were treated
by single intraperitoneal injection with 50 micrograms cadmium chloride
per kilogram body weight, a dose relevant to environmental exposures, and hepatic gene expression changes were determined at 1, 2, 4, 8, and
16 days postinjection in comparison to saline-treated controls. Gene
expression responses were confirmed by real-time reverse transcription
polymerase chain reaction (RT-PCR). Blast2GO gene ontology analysis
highlighted a general induction of the unfolded protein response, response to oxidative stress, protein synthesis, transport, and
degradation pathways, while apoptosis, cell cycle, cytoskeleton, and
cytokine genes were also affected. Transcript levels of cytochrome P450
1A (CYP1A) were repressed and vitellogenin altered, real-time PCR showed
induction of metallothionein. We thus describe the establishment of a
useful resource for ecotoxicogenomics and the determination of the
temporal molecular responses to cadmium, a prototypical heavy metal
pollutant.
11.
Conesa, A.; Nueda, M.; Ferrer, A.; Talon, M.
maSigPro: a method to identify significantly differential expression profiles in time-course microarray experiments Journal Article
In: BIOINFORMATICS, vol. 22, no. 9, pp. 1096-1102, 2006, ISSN: 1367-4803.
@article{ISI:000236997600009,
title = {maSigPro: a method to identify significantly differential expression
profiles in time-course microarray experiments},
author = { A. Conesa and M. Nueda and A. Ferrer and M. Talon},
url = {http://dx.doi.org/10.1093/bioinformatics/btl056},
doi = {10.1093/bioinformatics/btl056},
issn = {1367-4803},
year = {2006},
date = {2006-05-01},
journal = {BIOINFORMATICS},
volume = {22},
number = {9},
pages = {1096-1102},
abstract = {Motivation: Multi-series time-course microarray experiments are useful
approaches for exploring biological processes. In this type of
experiments, the researcher is frequently interested in studying gene
expression changes along time and in evaluating trend differences
between the various experimental groups. The large amount of data, multiplicity of experimental conditions and the dynamic nature of the
experiments poses great challenges to data analysis.
Results: In this work, we propose a statistical procedure to identify
genes that show different gene expression profiles across analytical
groups in time-course experiments. The method is a two-regression step
approach where the experimental groups are identified by dummy
variables. The procedure first adjusts a global regression model with
all the defined variables to identify differentially expressed genes, and in second a variable selection strategy is applied to study
differences between groups and to find statistically significant
different profiles. The methodology is illustrated on both a real and a
simulated microarray dataset.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Motivation: Multi-series time-course microarray experiments are useful
approaches for exploring biological processes. In this type of
experiments, the researcher is frequently interested in studying gene
expression changes along time and in evaluating trend differences
between the various experimental groups. The large amount of data, multiplicity of experimental conditions and the dynamic nature of the
experiments poses great challenges to data analysis.
Results: In this work, we propose a statistical procedure to identify
genes that show different gene expression profiles across analytical
groups in time-course experiments. The method is a two-regression step
approach where the experimental groups are identified by dummy
variables. The procedure first adjusts a global regression model with
all the defined variables to identify differentially expressed genes, and in second a variable selection strategy is applied to study
differences between groups and to find statistically significant
different profiles. The methodology is illustrated on both a real and a
simulated microarray dataset.
approaches for exploring biological processes. In this type of
experiments, the researcher is frequently interested in studying gene
expression changes along time and in evaluating trend differences
between the various experimental groups. The large amount of data, multiplicity of experimental conditions and the dynamic nature of the
experiments poses great challenges to data analysis.
Results: In this work, we propose a statistical procedure to identify
genes that show different gene expression profiles across analytical
groups in time-course experiments. The method is a two-regression step
approach where the experimental groups are identified by dummy
variables. The procedure first adjusts a global regression model with
all the defined variables to identify differentially expressed genes, and in second a variable selection strategy is applied to study
differences between groups and to find statistically significant
different profiles. The methodology is illustrated on both a real and a
simulated microarray dataset.
10.
Conesa, A.; Gotz, S.; Garcia-Gomez, J.; Terol, J.; Talon, M.; Robles, M.
Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research Journal Article
In: BIOINFORMATICS, vol. 21, no. 18, pp. 3674-3676, 2005, ISSN: 1367-4803.
@article{ISI:000231694600016,
title = {Blast2GO: a universal tool for annotation, visualization and analysis in
functional genomics research},
author = { A. Conesa and S. Gotz and J. Garcia-Gomez and J. Terol and M. Talon and M. Robles},
url = {http://dx.doi.org/10.1093/bioinformatics/bti610},
doi = {10.1093/bioinformatics/bti610},
issn = {1367-4803},
year = {2005},
date = {2005-09-01},
journal = {BIOINFORMATICS},
volume = {21},
number = {18},
pages = {3674-3676},
abstract = {We present here Blast2GO (B2G), a research tool designed with the main
purpose of enabling Gene Ontology (GO) based data mining on sequence
data for which no GO annotation is yet available. B2G joints in one
application GO annotation based on similarity searches with statistical
analysis and highlighted visualization on directed acyclic graphs. This
tool offers a suitable platform for functional genomics research in
non-model species. B2G is an intuitive and interactive desktop
application that allows monitoring and comprehension of the whole
annotation and analysis process.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We present here Blast2GO (B2G), a research tool designed with the main
purpose of enabling Gene Ontology (GO) based data mining on sequence
data for which no GO annotation is yet available. B2G joints in one
application GO annotation based on similarity searches with statistical
analysis and highlighted visualization on directed acyclic graphs. This
tool offers a suitable platform for functional genomics research in
non-model species. B2G is an intuitive and interactive desktop
application that allows monitoring and comprehension of the whole
annotation and analysis process.
purpose of enabling Gene Ontology (GO) based data mining on sequence
data for which no GO annotation is yet available. B2G joints in one
application GO annotation based on similarity searches with statistical
analysis and highlighted visualization on directed acyclic graphs. This
tool offers a suitable platform for functional genomics research in
non-model species. B2G is an intuitive and interactive desktop
application that allows monitoring and comprehension of the whole
annotation and analysis process.
9.
Forment, J.; Gadea, J.; Huerta, L.; Abizanda, L.; Agusti, J.; Alamar, S.; Alos, E.; Andres, F.; Arribas, R.; Beltran, J.; Berbel, A.; Blazquez, M.; Brumos, J.; Canas, L.; Cercos, M.; Colmenero-Flores, J.; Conesa, A.; Estables, B.; Gandia, M.; Garcia-Martinez, J.; Gimeno, J.; Gisbert, A.; Gomez, G.; Gonzalez-Candelas, L.; Granell, A.; Guerri, J.; Lafuente, M.; Madueno, F.; Marcos, J.; Marques, M.; Martinez, F.; Martinez-Godoy, M.; Miralles, S.; Moreno, P.; Navarro, L.; Pallas, V.; Perez-Amador, M.; Perez-Valle, J.; Pons, C.; Rodrigo, I.; Rodriguez, P.; Royo, C.; Serrano, R.; Soler, G.; Tadeo, F.; Talon, M.; Terol, J.; Trenor, M.; Vaello, L.; Vicente, O.; Vidal, C.; Zacarias, L.; Conejero, V.
Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies Journal Article
In: PLANT MOLECULAR BIOLOGY, vol. 57, no. 3, pp. 375-391, 2005, ISSN: 0167-4412.
@article{ISI:000229478400005,
title = {Development of a citrus genome-wide EST collection and cDNA microarray
as resources for genomic studies},
author = { J. Forment and J. Gadea and L. Huerta and L. Abizanda and J. Agusti and S. Alamar and E. Alos and F. Andres and R. Arribas and J. Beltran and A. Berbel and M. Blazquez and J. Brumos and L. Canas and M. Cercos and J. Colmenero-Flores and A. Conesa and B. Estables and M. Gandia and J. Garcia-Martinez and J. Gimeno and A. Gisbert and G. Gomez and L. Gonzalez-Candelas and A. Granell and J. Guerri and M. Lafuente and F. Madueno and J. Marcos and M. Marques and F. Martinez and M. Martinez-Godoy and S. Miralles and P. Moreno and L. Navarro and V. Pallas and M. Perez-Amador and J. Perez-Valle and C. Pons and I. Rodrigo and P. Rodriguez and C. Royo and R. Serrano and G. Soler and F. Tadeo and M. Talon and J. Terol and M. Trenor and L. Vaello and O. Vicente and C. Vidal and L. Zacarias and V. Conejero},
url = {http://dx.doi.org/10.1007/s11103-004-7926-1},
doi = {10.1007/s11103-004-7926-1},
issn = {0167-4412},
year = {2005},
date = {2005-02-01},
journal = {PLANT MOLECULAR BIOLOGY},
volume = {57},
number = {3},
pages = {375-391},
abstract = {A functional genomics project has been initiated to approach the
molecular characterization of the main biological and agronomical traits
of citrus. As a key part of this project, a citrus EST collection has
been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a
total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in
the citrus genome. Functional annotation of unigenes which have
Arabidopsis orthologues (68% of all unigenes) revealed gene
representation in every major functional category, suggesting that a
genome-wide EST collection was obtained. A Citrus clementina Hort. ex
Tan. cv. Clemenules genomic library, that will contribute to further
characterization of relevant genes, has also been constructed. To
initiate the analysis of citrus transcriptome, we have developed a cDNA
microarray containing 12,672 probes corresponding to 6875 putative
unigenes of the collection. Technical characterization of the microarray
showed high intra- and inter-array reproducibility, as well as a good
range of sensitivity. We have also validated gene expression data
achieved with this microarray through an independent technique such as
RNA gel blot analysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A functional genomics project has been initiated to approach the
molecular characterization of the main biological and agronomical traits
of citrus. As a key part of this project, a citrus EST collection has
been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a
total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in
the citrus genome. Functional annotation of unigenes which have
Arabidopsis orthologues (68% of all unigenes) revealed gene
representation in every major functional category, suggesting that a
genome-wide EST collection was obtained. A Citrus clementina Hort. ex
Tan. cv. Clemenules genomic library, that will contribute to further
characterization of relevant genes, has also been constructed. To
initiate the analysis of citrus transcriptome, we have developed a cDNA
microarray containing 12,672 probes corresponding to 6875 putative
unigenes of the collection. Technical characterization of the microarray
showed high intra- and inter-array reproducibility, as well as a good
range of sensitivity. We have also validated gene expression data
achieved with this microarray through an independent technique such as
RNA gel blot analysis.
molecular characterization of the main biological and agronomical traits
of citrus. As a key part of this project, a citrus EST collection has
been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a
total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in
the citrus genome. Functional annotation of unigenes which have
Arabidopsis orthologues (68% of all unigenes) revealed gene
representation in every major functional category, suggesting that a
genome-wide EST collection was obtained. A Citrus clementina Hort. ex
Tan. cv. Clemenules genomic library, that will contribute to further
characterization of relevant genes, has also been constructed. To
initiate the analysis of citrus transcriptome, we have developed a cDNA
microarray containing 12,672 probes corresponding to 6875 putative
unigenes of the collection. Technical characterization of the microarray
showed high intra- and inter-array reproducibility, as well as a good
range of sensitivity. We have also validated gene expression data
achieved with this microarray through an independent technique such as
RNA gel blot analysis.
8.
Yi, X.; Conesa, A.; Punt, P.; Hager, L.
Examining the role of glutamic acid 183 in chloroperoxidase catalysis Journal Article
In: JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 16, pp. 13855-13859, 2003, ISSN: 0021-9258.
@article{ISI:000182405000038,
title = {Examining the role of glutamic acid 183 in chloroperoxidase catalysis},
author = { X. Yi and A. Conesa and P. Punt and L. Hager},
url = {http://dx.doi.org/10.1074/jbc.M210906200},
doi = {10.1074/jbc.M210906200},
issn = {0021-9258},
year = {2003},
date = {2003-04-01},
journal = {JOURNAL OF BIOLOGICAL CHEMISTRY},
volume = {278},
number = {16},
pages = {13855-13859},
abstract = {Site-directed mutagenesis has been used to investigate the role of
glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray
crystallographic structure of chloroperoxidase, Glu-183 is postulated to
function on distal side of the heme prosthetic group as an acid-base
catalyst in facilitating the reaction between the peroxidase and
hydrogen peroxide with the formation of Compound I. In contrast, the
other members of the heme peroxidase family use a histidine residue in
this role. Plasmids have now been constructed in which the codon for
Glu-183 is replaced with a histidine codon. The mutant recombinant gene
has been expressed in Aspergillus niger. An analysis of the produced
mutant gene shows that the substitution of Glu-183 with a His residue is
detrimental to the chlorination and dismutation activity of
chloroperoxidase. The activity is reduced by 85 and 50% of wild type
activity, respectively. However, quite unexpectedly, the epoxidation
activity of the mutant enzyme is significantly enhanced similar
to2.5-fold. These results show that Glu-183 is important but not
essential for the chlorination activity of chloroperoxidase. It is
possible that the increased epoxidation of the mutant enzyme is based on
an increase in the hydrophobicity of the active site.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Site-directed mutagenesis has been used to investigate the role of
glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray
crystallographic structure of chloroperoxidase, Glu-183 is postulated to
function on distal side of the heme prosthetic group as an acid-base
catalyst in facilitating the reaction between the peroxidase and
hydrogen peroxide with the formation of Compound I. In contrast, the
other members of the heme peroxidase family use a histidine residue in
this role. Plasmids have now been constructed in which the codon for
Glu-183 is replaced with a histidine codon. The mutant recombinant gene
has been expressed in Aspergillus niger. An analysis of the produced
mutant gene shows that the substitution of Glu-183 with a His residue is
detrimental to the chlorination and dismutation activity of
chloroperoxidase. The activity is reduced by 85 and 50% of wild type
activity, respectively. However, quite unexpectedly, the epoxidation
activity of the mutant enzyme is significantly enhanced similar
to2.5-fold. These results show that Glu-183 is important but not
essential for the chlorination activity of chloroperoxidase. It is
possible that the increased epoxidation of the mutant enzyme is based on
an increase in the hydrophobicity of the active site.
glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray
crystallographic structure of chloroperoxidase, Glu-183 is postulated to
function on distal side of the heme prosthetic group as an acid-base
catalyst in facilitating the reaction between the peroxidase and
hydrogen peroxide with the formation of Compound I. In contrast, the
other members of the heme peroxidase family use a histidine residue in
this role. Plasmids have now been constructed in which the codon for
Glu-183 is replaced with a histidine codon. The mutant recombinant gene
has been expressed in Aspergillus niger. An analysis of the produced
mutant gene shows that the substitution of Glu-183 with a His residue is
detrimental to the chlorination and dismutation activity of
chloroperoxidase. The activity is reduced by 85 and 50% of wild type
activity, respectively. However, quite unexpectedly, the epoxidation
activity of the mutant enzyme is significantly enhanced similar
to2.5-fold. These results show that Glu-183 is important but not
essential for the chlorination activity of chloroperoxidase. It is
possible that the increased epoxidation of the mutant enzyme is based on
an increase in the hydrophobicity of the active site.
7.
Punt, P.; van Biezen, N.; Conesa, A.; Albers, A.; Mangnus, J.; van den Hondel, C.
Filamentous fungi as cell factories for heterologous protein production Journal Article
In: TRENDS IN BIOTECHNOLOGY, vol. 20, no. 5, pp. 200-206, 2002, ISSN: 0167-7799.
@article{ISI:000175065300008,
title = {Filamentous fungi as cell factories for heterologous protein production},
author = { P. Punt and N. van Biezen and A. Conesa and A. Albers and J. Mangnus and C. van den Hondel},
url = {http://dx.doi.org/10.1016/S0167-7799(02)01933-9},
doi = {10.1016/S0167-7799(02)01933-9},
issn = {0167-7799},
year = {2002},
date = {2002-05-01},
journal = {TRENDS IN BIOTECHNOLOGY},
volume = {20},
number = {5},
pages = {200-206},
abstract = {Filamentous fungi have been used as sources of metabolites and enzymes
for centuries. For about two decades, molecular genetic tools have
enabled us to use these organisms to express extra copies of both
endogenous and exogenous genes. This review of current practice reveals
that molecular tools have enabled several new developments. But it has
been process development that has driven the final breakthrough to
achieving commercially relevant quantities of protein. Recent research
into gene expression in filamentous fungi has explored their wealth of
genetic diversity with a view to exploiting them as expression hosts and
as a source of new genes. Inevitably, the progress in the `genomics'
technology will further develop high-throughput technologies for these
organisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Filamentous fungi have been used as sources of metabolites and enzymes
for centuries. For about two decades, molecular genetic tools have
enabled us to use these organisms to express extra copies of both
endogenous and exogenous genes. This review of current practice reveals
that molecular tools have enabled several new developments. But it has
been process development that has driven the final breakthrough to
achieving commercially relevant quantities of protein. Recent research
into gene expression in filamentous fungi has explored their wealth of
genetic diversity with a view to exploiting them as expression hosts and
as a source of new genes. Inevitably, the progress in the `genomics'
technology will further develop high-throughput technologies for these
organisms.
for centuries. For about two decades, molecular genetic tools have
enabled us to use these organisms to express extra copies of both
endogenous and exogenous genes. This review of current practice reveals
that molecular tools have enabled several new developments. But it has
been process development that has driven the final breakthrough to
achieving commercially relevant quantities of protein. Recent research
into gene expression in filamentous fungi has explored their wealth of
genetic diversity with a view to exploiting them as expression hosts and
as a source of new genes. Inevitably, the progress in the `genomics'
technology will further develop high-throughput technologies for these
organisms.
6.
Conesa, A.; Punt, P.; van den Hondel, C.
Fungal peroxidases: molecular aspects and applications Journal Article
In: JOURNAL OF BIOTECHNOLOGY, vol. 93, no. 2, pp. 143-158, 2002, ISSN: 0168-1656.
@article{ISI:000173535800005,
title = {Fungal peroxidases: molecular aspects and applications},
author = { A. Conesa and P. Punt and C. van den Hondel},
url = {http://dx.doi.org/10.1016/S0168-1656(01)00394-7},
doi = {10.1016/S0168-1656(01)00394-7},
issn = {0168-1656},
year = {2002},
date = {2002-02-01},
journal = {JOURNAL OF BIOTECHNOLOGY},
volume = {93},
number = {2},
pages = {143-158},
abstract = {Peroxidases are oxidoreductases that utilize hydrogen peroxide to
catalyze oxidative reactions. A large number of peroxidases have been
identified in fungal species and are being characterized at the
molecular level. In this manuscript we review the current knowledge on
the molecular aspects of this type of enzymes. We present an overview of
the research efforts undertaken in deciphering the structural basis of
the catalytic properties of fungal peroxidases and discuss molecular
genetics and protein homology aspects of this enzyme class. Finally, we
summarize the potential biotechnological applications of these enzymes
and evaluate recent advances on their expression in heterologous systems
for production purposes. (C) 2002 Elsevier Science B.V. All rights
reserved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Peroxidases are oxidoreductases that utilize hydrogen peroxide to
catalyze oxidative reactions. A large number of peroxidases have been
identified in fungal species and are being characterized at the
molecular level. In this manuscript we review the current knowledge on
the molecular aspects of this type of enzymes. We present an overview of
the research efforts undertaken in deciphering the structural basis of
the catalytic properties of fungal peroxidases and discuss molecular
genetics and protein homology aspects of this enzyme class. Finally, we
summarize the potential biotechnological applications of these enzymes
and evaluate recent advances on their expression in heterologous systems
for production purposes. (C) 2002 Elsevier Science B.V. All rights
reserved.
catalyze oxidative reactions. A large number of peroxidases have been
identified in fungal species and are being characterized at the
molecular level. In this manuscript we review the current knowledge on
the molecular aspects of this type of enzymes. We present an overview of
the research efforts undertaken in deciphering the structural basis of
the catalytic properties of fungal peroxidases and discuss molecular
genetics and protein homology aspects of this enzyme class. Finally, we
summarize the potential biotechnological applications of these enzymes
and evaluate recent advances on their expression in heterologous systems
for production purposes. (C) 2002 Elsevier Science B.V. All rights
reserved.
5.
Conesa, A.; Jeenes, D.; Archer, D.; van den Hondel, C.; Punt, P.
Calnexin overexpression increases manganese peroxidase production in Aspergillus niger Journal Article
In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 68, no. 2, pp. 846-851, 2002, ISSN: 0099-2240.
@article{ISI:000173588600051,
title = {Calnexin overexpression increases manganese peroxidase production in
Aspergillus niger},
author = { A. Conesa and D. Jeenes and D. Archer and C. van den Hondel and P. Punt},
url = {http://dx.doi.org/10.1128/AEM.68.2.846-851.2002},
doi = {10.1128/AEM.68.2.846-851.2002},
issn = {0099-2240},
year = {2002},
date = {2002-02-01},
journal = {APPLIED AND ENVIRONMENTAL MICROBIOLOGY},
volume = {68},
number = {2},
pages = {846-851},
abstract = {Heme-containing peroxidases from white rot basidiomycetes, in contrast
to most proteins of fungal origin, are poorly produced in industrial
filamentous fungal strains. Factors limiting peroxidase production are
believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for
peroxidase biosynthesis has been proposed to be an important bottleneck.
In this work, we analyzed the role of two components of the secretion
pathway, the chaperones calnexin and binding protein (BiP), in the
production of a fungal peroxidase. Expression of the Phanerochaete
chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted
in an increase in the expression level of the clxA and bipA genes. In a
heme-supplemented medium, where MnP was shown to be overproduced to
higher levels, induction of clxA and bipA was also higher.
Overexpression of these two chaperones in an MnP-producing strain was
analyzed for its effect on MnP production. Whereas bipA overexpression
seriously reduced MnP production, overexpression of calnexin resulted in
a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin
overexpression had no synergistic effect on MnP production. The possible
function of these two chaperones in MnP maturation and production is
discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Heme-containing peroxidases from white rot basidiomycetes, in contrast
to most proteins of fungal origin, are poorly produced in industrial
filamentous fungal strains. Factors limiting peroxidase production are
believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for
peroxidase biosynthesis has been proposed to be an important bottleneck.
In this work, we analyzed the role of two components of the secretion
pathway, the chaperones calnexin and binding protein (BiP), in the
production of a fungal peroxidase. Expression of the Phanerochaete
chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted
in an increase in the expression level of the clxA and bipA genes. In a
heme-supplemented medium, where MnP was shown to be overproduced to
higher levels, induction of clxA and bipA was also higher.
Overexpression of these two chaperones in an MnP-producing strain was
analyzed for its effect on MnP production. Whereas bipA overexpression
seriously reduced MnP production, overexpression of calnexin resulted in
a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin
overexpression had no synergistic effect on MnP production. The possible
function of these two chaperones in MnP maturation and production is
discussed.
to most proteins of fungal origin, are poorly produced in industrial
filamentous fungal strains. Factors limiting peroxidase production are
believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for
peroxidase biosynthesis has been proposed to be an important bottleneck.
In this work, we analyzed the role of two components of the secretion
pathway, the chaperones calnexin and binding protein (BiP), in the
production of a fungal peroxidase. Expression of the Phanerochaete
chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted
in an increase in the expression level of the clxA and bipA genes. In a
heme-supplemented medium, where MnP was shown to be overproduced to
higher levels, induction of clxA and bipA was also higher.
Overexpression of these two chaperones in an MnP-producing strain was
analyzed for its effect on MnP production. Whereas bipA overexpression
seriously reduced MnP production, overexpression of calnexin resulted in
a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin
overexpression had no synergistic effect on MnP production. The possible
function of these two chaperones in MnP maturation and production is
discussed.
4.
Conesa, A.; Punt, P.; van Luijk, N.; van den Hondel, C.
The secretion pathway in filamentous fungi: A biotechnological view Journal Article
In: FUNGAL GENETICS AND BIOLOGY, vol. 33, no. 3, pp. 155-171, 2001, ISSN: 1087-1845.
@article{ISI:000170506100001,
title = {The secretion pathway in filamentous fungi: A biotechnological view},
author = { A. Conesa and P. Punt and N. van Luijk and C. van den Hondel},
url = {http://dx.doi.org/10.1006/fgbe.2001.1276},
doi = {10.1006/fgbe.2001.1276},
issn = {1087-1845},
year = {2001},
date = {2001-08-01},
journal = {FUNGAL GENETICS AND BIOLOGY},
volume = {33},
number = {3},
pages = {155-171},
abstract = {The high capacity of the secretion machinery of filamentous fungi has
been widely exploited for the production of homologous and heterologous
proteins; however, our knowledge of the fungal secretion pathway is
still at an early stage. Most of the knowledge comes from models
developed in yeast and higher eukaryotes, which have served as reference
for the studies on fungal species. In this review we compile the data
accumulated in recent years on the molecular basis of fungal secretion, emphasizing the relevance of these data for the biotechnological use of
the fungal cell and indicating how this information has been applied in
attempts to create improved production strains. We also present recent
emerging approaches that promise to provide answers to fundamental
questions on the molecular genetics of the fungal secretory pathway. (C)
2001 Academic Press.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The high capacity of the secretion machinery of filamentous fungi has
been widely exploited for the production of homologous and heterologous
proteins; however, our knowledge of the fungal secretion pathway is
still at an early stage. Most of the knowledge comes from models
developed in yeast and higher eukaryotes, which have served as reference
for the studies on fungal species. In this review we compile the data
accumulated in recent years on the molecular basis of fungal secretion, emphasizing the relevance of these data for the biotechnological use of
the fungal cell and indicating how this information has been applied in
attempts to create improved production strains. We also present recent
emerging approaches that promise to provide answers to fundamental
questions on the molecular genetics of the fungal secretory pathway. (C)
2001 Academic Press.
been widely exploited for the production of homologous and heterologous
proteins; however, our knowledge of the fungal secretion pathway is
still at an early stage. Most of the knowledge comes from models
developed in yeast and higher eukaryotes, which have served as reference
for the studies on fungal species. In this review we compile the data
accumulated in recent years on the molecular basis of fungal secretion, emphasizing the relevance of these data for the biotechnological use of
the fungal cell and indicating how this information has been applied in
attempts to create improved production strains. We also present recent
emerging approaches that promise to provide answers to fundamental
questions on the molecular genetics of the fungal secretory pathway. (C)
2001 Academic Press.
3.
Conesa, A.; Weelink, G.; van den Hondel, C.; Punt, P.
C-terminal propeptide of the Caldariomyces fumago chloroperoxidase: an intramolecular chaperone? Journal Article
In: FEBS LETTERS, vol. 503, no. 2-3, pp. 117-120, 2001, ISSN: 0014-5793.
@article{ISI:000170641000001,
title = {C-terminal propeptide of the Caldariomyces fumago chloroperoxidase: an
intramolecular chaperone?},
author = { A. Conesa and G. Weelink and C. van den Hondel and P. Punt},
url = {http://dx.doi.org/10.1016/S0014-5793(01)02698-9},
doi = {10.1016/S0014-5793(01)02698-9},
issn = {0014-5793},
year = {2001},
date = {2001-08-01},
journal = {FEBS LETTERS},
volume = {503},
number = {2-3},
pages = {117-120},
abstract = {The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a
372-aa precursor which undergoes two proteolytic processing events.
removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal
propeptide. The Aspergillus niger expression system developed for CPO
was used to get insight into the function of this C-terminal propeptide.
A. niger transformants expressing a CPO protein from which the
C-terminal propeptide was deleted failed in producing any extracellular
CPO activity, although the CPO polypeptide was synthesised. Expression
of the full-length gene in an A. niger strain lacking the KEX2-like
protease PclA also resulted in the production of CPO cross-reactive
material into the culture medium, but no CPO activity. Based on these
results, a function of the C-terminal propeptide in CPO maturation is
indicated. (C) 2001 Federation of European Biochemical Societies.
Published by Elsevier Science B.V. All rights reserved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a
372-aa precursor which undergoes two proteolytic processing events.
removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal
propeptide. The Aspergillus niger expression system developed for CPO
was used to get insight into the function of this C-terminal propeptide.
A. niger transformants expressing a CPO protein from which the
C-terminal propeptide was deleted failed in producing any extracellular
CPO activity, although the CPO polypeptide was synthesised. Expression
of the full-length gene in an A. niger strain lacking the KEX2-like
protease PclA also resulted in the production of CPO cross-reactive
material into the culture medium, but no CPO activity. Based on these
results, a function of the C-terminal propeptide in CPO maturation is
indicated. (C) 2001 Federation of European Biochemical Societies.
Published by Elsevier Science B.V. All rights reserved.
372-aa precursor which undergoes two proteolytic processing events.
removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal
propeptide. The Aspergillus niger expression system developed for CPO
was used to get insight into the function of this C-terminal propeptide.
A. niger transformants expressing a CPO protein from which the
C-terminal propeptide was deleted failed in producing any extracellular
CPO activity, although the CPO polypeptide was synthesised. Expression
of the full-length gene in an A. niger strain lacking the KEX2-like
protease PclA also resulted in the production of CPO cross-reactive
material into the culture medium, but no CPO activity. Based on these
results, a function of the C-terminal propeptide in CPO maturation is
indicated. (C) 2001 Federation of European Biochemical Societies.
Published by Elsevier Science B.V. All rights reserved.
2.
Conesa, A.; van de Velde, F.; van Rantwijk, F.; Sheldon, R.; van den Hondel, C.; Punt, P.
Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme Journal Article
In: JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 21, pp. 17635-17640, 2001, ISSN: 0021-9258.
@article{ISI:000168866500004,
title = {Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus
niger and characterization of the recombinant enzyme},
author = { A. Conesa and F. van de Velde and F. van Rantwijk and R. Sheldon and C. van den Hondel and P. Punt},
url = {http://dx.doi.org/10.1074/jbc.M010571200},
doi = {10.1074/jbc.M010571200},
issn = {0021-9258},
year = {2001},
date = {2001-05-01},
journal = {JOURNAL OF BIOLOGICAL CHEMISTRY},
volume = {276},
number = {21},
pages = {17635-17640},
abstract = {The Caldariomyces fumago chloroperoxidase was successfully expressed in
Aspergillus niger. The recombinant enzyme was produced in the culture
medium as an active protein and could be purified by a three-step
purification procedure. The catalytic behavior of recombinant
chloroperoxidase (rCPO) was studied and compared with that of native
CPO. The specific chlorination activity (47 units/nmol) of rCPO and its
pH optimum (pH 2.75) were very similar to those of native CPO. rCPO
catalyzes the oxidation of various substrates in comparable yields and
selectivities to native CPO. Indole was oxidized to 2-oxindole with 99%
selectivity and thioanisole to the corresponding R-sulfoxide
(enantiomeric excess >98%). Incorporation of O-18 from labeled
(H2O2)-O-18 into the oxidized products was 100% in both cases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The Caldariomyces fumago chloroperoxidase was successfully expressed in
Aspergillus niger. The recombinant enzyme was produced in the culture
medium as an active protein and could be purified by a three-step
purification procedure. The catalytic behavior of recombinant
chloroperoxidase (rCPO) was studied and compared with that of native
CPO. The specific chlorination activity (47 units/nmol) of rCPO and its
pH optimum (pH 2.75) were very similar to those of native CPO. rCPO
catalyzes the oxidation of various substrates in comparable yields and
selectivities to native CPO. Indole was oxidized to 2-oxindole with 99%
selectivity and thioanisole to the corresponding R-sulfoxide
(enantiomeric excess >98%). Incorporation of O-18 from labeled
(H2O2)-O-18 into the oxidized products was 100% in both cases.
Aspergillus niger. The recombinant enzyme was produced in the culture
medium as an active protein and could be purified by a three-step
purification procedure. The catalytic behavior of recombinant
chloroperoxidase (rCPO) was studied and compared with that of native
CPO. The specific chlorination activity (47 units/nmol) of rCPO and its
pH optimum (pH 2.75) were very similar to those of native CPO. rCPO
catalyzes the oxidation of various substrates in comparable yields and
selectivities to native CPO. Indole was oxidized to 2-oxindole with 99%
selectivity and thioanisole to the corresponding R-sulfoxide
(enantiomeric excess >98%). Incorporation of O-18 from labeled
(H2O2)-O-18 into the oxidized products was 100% in both cases.
1.
Conesa, A.; van den Hondel, C.; Punt, P.
Studies on the production of fungal peroxidases in Aspergillus niger Journal Article
In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 66, no. 7, pp. 3016-3023, 2000, ISSN: 0099-2240.
@article{ISI:000088057600044,
title = {Studies on the production of fungal peroxidases in Aspergillus niger},
author = { A. Conesa and C. van den Hondel and P. Punt},
url = {http://dx.doi.org/10.1128/AEM.66.7.3016-3023.2000},
doi = {10.1128/AEM.66.7.3016-3023.2000},
issn = {0099-2240},
year = {2000},
date = {2000-07-01},
journal = {APPLIED AND ENVIRONMENTAL MICROBIOLOGY},
volume = {66},
number = {7},
pages = {3016-3023},
abstract = {To get insight into the limiting factors existing for the efficient
production of fungal peroxidase in filamentous fungi, the expression of
the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and
manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been
studied. For this purpose, a protease-deficient A. niger strain and
different expression cassettes have been used. Northern blotting
experiments indicated high steady-state mRNA levels for the recombinant
genes. Manganese peroxidase was secreted into the culture medium as an
active protein. The recombinant protein showed specific activity and a
spectrum profile similar to those of the native enzyme, was correctly
processed at its N terminus, and had a slightly lower mobility on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, Recombinant MnP
production could be increased up to 100 mg/liter upon hemoglobin
supplementation of the culture medium. Lignin peroxidase was also
secreted into the extracellular medium, although the protein was not
active, presumably due to incorrect processing of the secreted enzyme.
Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase
gene did not result in improved production yields.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To get insight into the limiting factors existing for the efficient
production of fungal peroxidase in filamentous fungi, the expression of
the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and
manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been
studied. For this purpose, a protease-deficient A. niger strain and
different expression cassettes have been used. Northern blotting
experiments indicated high steady-state mRNA levels for the recombinant
genes. Manganese peroxidase was secreted into the culture medium as an
active protein. The recombinant protein showed specific activity and a
spectrum profile similar to those of the native enzyme, was correctly
processed at its N terminus, and had a slightly lower mobility on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, Recombinant MnP
production could be increased up to 100 mg/liter upon hemoglobin
supplementation of the culture medium. Lignin peroxidase was also
secreted into the extracellular medium, although the protein was not
active, presumably due to incorrect processing of the secreted enzyme.
Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase
gene did not result in improved production yields.
production of fungal peroxidase in filamentous fungi, the expression of
the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and
manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been
studied. For this purpose, a protease-deficient A. niger strain and
different expression cassettes have been used. Northern blotting
experiments indicated high steady-state mRNA levels for the recombinant
genes. Manganese peroxidase was secreted into the culture medium as an
active protein. The recombinant protein showed specific activity and a
spectrum profile similar to those of the native enzyme, was correctly
processed at its N terminus, and had a slightly lower mobility on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, Recombinant MnP
production could be increased up to 100 mg/liter upon hemoglobin
supplementation of the culture medium. Lignin peroxidase was also
secreted into the extracellular medium, although the protein was not
active, presumably due to incorrect processing of the secreted enzyme.
Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase
gene did not result in improved production yields.